Hemoglobins with Altered Oxygen Affinity, Unstable Hemoglobins, M-Hemoglobins, and Dyshemoglobinemias
Martin H. Steinberg
More than 1,100 mutations affecting the globin subunits of hemoglobin have been described (http://globin.cse.psu.edu/), and among these, sickle cell disease along with hemoglobinopathies associated with HbE, HbC, and the β– and α-thalassemias are mankind’s most common single-gene disorders (see Chapters 33 and 34). Much less common are hemoglobin mutations discussed in this chapter that affect the ability of the molecule to bind and release oxygen, that reduce its stability, and that allow its heme iron to be oxidized. Exogenous agents can also oxidize hemoglobin, interfering with oxygen transport.
HEMOGLOBINS WITH ALTERED OXYGEN AFFINITY
The affinity of hemoglobin for oxygen is characterized by the amount of oxygen bound at any given oxygen tension. Oxygen affinity is usually designated by the P50, which is the partial pressure of oxygen at which hemoglobin is 50% saturated. Oxygen affinity can be modified by pH, temperature, and organic phosphates. The normal co-operativity of hemoglobin, or heme-heme interactions in the hemoglobin tetramer, determines the sigmoidal shape of the hemoglobin-oxygen dissociation curve. This is a result of the fact that the deoxygenated T (tense) form of hemoglobin has a lower affinity for oxygen than the oxygenated R (relaxed) form (see Chapter 6). Consequently, globin gene mutations that alter areas of the molecule involved with T-R interactions can lead to alterations in oxygen affinity. Both high- and low-oxygen-affinity hemoglobin variants are encountered and all globin genes have been affected.
High-oxygen-Affinity Hemoglobins
Globin gene mutations can increase the affinity of the hemoglobin molecule for oxygen and cause erythrocytosis. More than 90 high-oxygen-affinity variants have been reported in http://globin.cse.psu.edu/ and Wajcman and Galacteros.1 Familial erythrocytosis is a valuable clue to the presence of a high-oxygen-affinity hemoglobin variant; isolated cases are caused by new mutations. These α– or β-globin chain mutations are dominant disorders, expressed clinically in the heterozygote. They may be lethal in homozygotes if they affect the α-globin chain and are expressed in utero. One γ-globin variant (HbF-Monserrato-Sassari, HBG2 cys93arg) in a normal newborn and a possible δ-globin variant (Hb Noah Mehmet Oesteurk, HBD his143tyr) have been described.
Several homozygotes for β-globin high-oxygen-affinity variants have been described and these individuals can have more severe disease.2, 3, 4, 5 Compound heterozygotes with a high-oxygen-affinity hemoglobin and β0-thalassemia have also been described, mimicking the homozygous state as normal adult hemoglobin A (HbA) is not present.
Pathophysiology
Stabilization of the R state of the hemoglobin tetramer (α2β2), with its high affinity for oxygen, or destabilization of the low-oxygen-affinity T state is caused by globin gene mutations in critical areas that can effect the R→T transition (Table 35.1).6 The increased avidity for oxygen (low P50) of these variants reduces oxygen delivery to tissues, thereby stimulating erythropoietin production and increasing red cell mass. Patients with high-oxygen-affinity hemoglobins with erythrocytosis usually have normal urine erythropoietin levels. However, erythropoietin levels increase when they are phlebotomized to a normal red cell mass.7, 8 Individuals with these hemoglobin variants appear to be reasonably compensated for the low P50 by the increased red cell mass. Oxygen consumption and arterial pO2 are normal, but in some cases there is reduced mixed venous pO2 and decreased resting cardiac output.
Not unexpectedly, the JAK2 V617F mutation found in some individuals with polycythemia vera is absent in patients with high-oxygen-affinity hemoglobin variant induced erythropoiesis.9
Diagnosis
The differential diagnosis of increased red cell mass is discussed in Chapter 44. The diagnosis of high-oxygen-affinity hemoglobins is suspected by finding isolated erythrocytosis without accompanying leukocytosis, thrombocytosis, and splenomegaly, as might be expected in cases of polycythemia vera. A family history of erythrocytosis is also suggestive of a high-oxygen-affinity hemoglobin variant. Other disorders expressed in the erythrocyte that cause isolated erythrocytosis include 2,3-diphosphoglycerate (2,3-DPG) mutase mutations that reduce the synthesis of this modulator of hemoglobin-oxygen affinity, some instances of methemoglobinemia, and chronic carbon monoxide (CO) poisoning.
Determination of the red cell oxygen equilibrium curve is the benchmark for the diagnosis of erythrocytosis due to highoxygen-affinity hemoglobins. The oxygen-binding characteristics of hybrid tetramers (α2βAβvar) are likely to be intermediate between purified HbA and purified variant, and the shape of the hemoglobin-oxygen dissociation curve can at times be biphasic. Measurement of blood P50 confirms the shift in the hemoglobin-oxygen dissociation curve. Rarely, the whole-blood P50 is normal, requiring study of dialyzed purified hemoglobin. An accurate P50 value is difficult to “calculate” from pO2 data, and it is best to measure the pO2 and hemoglobin saturation directly. P50 measurements are not widely available but can be done with several instruments.
High oxygen affinity is observed in both erythrocytes and purified dialyzed hemoglobin. The concentration of 2,3-DPG is normal, indicating that altered oxygen affinity is not caused by reduced levels of this modulator of hemoglobin function. This is in contrast to what is seen in the rare instance of red cell 2,3-DPG mutase deficiency, where both erythrocytes and whole blood have high oxygen affinity, and the oxygen affinity of the purified hemolysate stripped of 2,3-DPG is normal.10
High-performance liquid chromatography (HPLC) studies may reveal an abnormal hemoglobin, but normal studies do not exclude the possibility of a high-affinity hemoglobin that migrates with normal HbA. As with all evaluations for abnormal hemoglobins, determining the DNA sequence of the globin genes provides the definitive information.
Examples of high-oxygen-affinity hemoglobins that illustrate the varying mechanisms and heterogeneous clinical findings seen with these variants are discussed in the following. A detailed
understanding of the structure of hemoglobin and the importance of each residue in determining function allows a molecular explanation of most of the clinical abnormalities observed.
understanding of the structure of hemoglobin and the importance of each residue in determining function allows a molecular explanation of most of the clinical abnormalities observed.
TABLE 35.1 SITES OF GLOBIN MUTATION ASSOCIATED WITH INCREASED OXYGEN AFFINITYa | ||||||
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α-Globin Chain Variants
Because there are four α-globin genes, most stable α-globin variants form 25% or less of the total hemoglobin, compared with 40% to 50% for β-globin variants. As a result, the clinical effects of α-globin variants are less striking than those of β-globin variants. However, the coincident inheritance of a β-thalassemia gene can modulate the concentration of high-affinity α-variant hemoglobins and homozygosity for a high-oxygen-affinity variant affecting α-globin chains has been described.
Hb Chesapeake (HBA arg92leu) was the first reported highaffinity hemoglobin variant.11 It was discovered in an 81-year-old patient with erythrocytosis, an abnormal hemoglobin detected by hemoglobin electrophoresis, and erythrocytes with increased oxygen affinity. Fifteen members of the proband’s family were similarly affected. Hb Chesapeake represented ˜20% of the total hemoglobin. With a P50 of 19 mm Hg (normal ˜26 mm Hg), Hb Chesapeake produced moderate erythrocytosis. The mutation affected an invariant residue that stabilizes the R state at the α1β2 area of contact, making the T conformer less favored.
Hb Nunobiki (HBA1 arg141cys), is one of four mutations of this invariant residue, all of which exhibit high oxygen affinity and moderate to mild erythrocytosis.12 This group of mutations represents an interesting cluster of variants that illustrates the effects of different mutations at the same amino acid residue. As a mutant of the 3′-HBA1 gene that is expressed to a lesser extent than the 5′-HBA2 gene, Hb Nunobiki makes up ˜13% of the hemolysate and is accompanied by only mild erythrocytosis. High oxygen affinity is a result of the breaking of the C terminal-to-C terminal salt bridge that is indispensable for the stabilization of the T state, favoring the R state.
β-Globin Chain Variants
All possible single-base mutations of the β99 site disturbing the α1β2 area of contact have been described and include Hb Kempsey (HBB asp99asn), Hb Yakima (asp99his), Hb Radcliffe (asp99ala), Hb Ypsilanti (asp99tyr), Hb Hotel-Dieu (asp99gly), Hb Chemilly (asp99val), and Hb Coimbra (asp99glu). As expected for stable β-globin chain variants, all are present at 40% to 50% of the hemolysate, exhibit moderately high oxygen affinity, and are characterized clinically by erythrocytosis. Hbs Kempsey, Radcliffe, and Hotel Dieu have a decreased response to 2,3-DPG. Hbs Ypsilanti and Radcliffe form stable hybrid tetramers in the hemolysates in which the abnormal β chains coexist with normal β chains.
Six of the possible seven mutations of the C-terminal CAC (tyr) codon have also been described. One of them, Hb Cochin-Port Royal (tyr146arg), has nearly normal oxygen affinity but decreased 2,3-DPG interaction and Bohr effect.13
Three mutations of β82 lys have been described: Hb Rahere (lys82thr), Hb Helsinki (lys82met), and Hb Providence (lys82asn). All have moderately high oxygen affinity and moderate erythrocytosis.
These mutants have drastically reduced 2,3-DPG binding as a result of the elimination of one of the normal binding sites for this allosteric effector. Hb Porto Alegre (HBB ser9cys) has high oxygen affinity and a tendency to aggregate, but erythrocytosis is not present.14 Polymerization of Hb Porto Alegre is based on the formation of disulfide bonds in oxygenated samples and is different from HbS polymerization. Polymerization of this mutant diminishes heme-heme interaction and increases the oxygen affinity.
Hb Tak (HBB 147(+AC), modified C-terminal sequence: 147thr-lys-leu-ala-phe-leu-leu-ser-asn-phe-157tyr-COOH), is elongated by 11 amino acid residues.15, 16 It forms 40% of the hemolysate, has a very high oxygen affinity with no co-operativity, and no allosteric interaction with pH or 2,3-DPG. The C terminus of the β-globin chain is actively involved in the conformational changes of the hemoglobin molecule by stabilizing the T state. By having these stabilizing interactions disrupted, Hb Tak is totally frozen in the R state. Hb Tak is also slightly unstable. In spite of these severe functional abnormalities, a heterozygous patient did not have erythrocytosis. The extreme biphasic nature of the hemoglobin-oxygen affinity curve observed in mixtures of Hb Tak and HbA suggests that hybrid tetramer (α2βAβTak) formation is absent. The top portion of the oxygen equilibrium curve is normal, and it begins to be abnormal only at <40% saturation. Because physiologic oxygen exchange occurs most commonly above that level of saturation, the tissues may not be hypoxic, removing the stimulus for increased erythropoiesis.
Clinical Features
Patients with high-affinity hemoglobins and erythrocytosis have a benign clinical course and rarely have complications, apart from a ruddy complexion. Splenomegaly is typically absent. Hemoglobin concentration and hematocrit are increased variably, and usually only moderately, suggesting that modulation by variations in other genes might affect the physiologic response to hypoxia. Some patients with Hb Malmo (HBB his97gln) have been reported to be symptomatic and to benefit from phlebotomy and the transfusion of normal blood, but this clinical course is an exception.17
Many cases of high-oxygen-affinity hemoglobins are diagnosed during a routine hematologic examination or when the family of a proband known to have erythrocytosis is examined. In very limited studies, exercise capacity in the laboratory and the indices of working capacity and cardiac tolerance were similar in patients with high-oxygen-affinity hemoglobins and in controls.18 It has been suspected that carriers of these variants may have enhanced athletic performance under some circumstances, and this has led to the unfortunate and sometimes fatal use of erythropoietin or transfusion to enhance performance in competitive athletics.
In a population-based study of erythrocytosis, high-oxygen-affinity variants accounted for 3% of all cases.19 By early diagnosis of high-affinity hemoglobins, unnecessary invasive diagnostic procedures and inappropriate therapeutic interventions, such as cardiac catheterization, can be avoided. Patients have received 32P treatment based on a mistaken diagnosis of polycythemia vera.
Increased morbidity or mortality in mothers with high-oxygen-affinity hemoglobins or their offspring has not been observed, suggesting that the affinity of the mother’s hemoglobin is irrelevant with respect to oxygen delivery to the fetus.18 Low ambient pO2, as in unpressurized airplanes and ascent to altitude, do not represent a risk, because high-affinity hemoglobins are avid for oxygen.
Hypothetically, carriers should be less prone to “the bends” during deep sea diving, because of slower oxygen release during ascension.
Treatment
Patients with high-oxygen-affinity hemoglobins have reasonable compensation for their abnormality, with adequate tissue oxygen delivery despite increases in blood viscosity. Intervention is therefore rarely required. Exercise studies before and after phlebotomy in patients with Hb Osler (HBB tyr145asn), a variant with a P50 of 10 to 11 mm Hg and a hemoglobin concentration of ˜22 g/dl, did not show an impairment after phlebotomy.18 Limited studies have suggested that phlebotomy generally does not improve exercise performance. However, rare individuals appear to have benefited from phlebotomy, and thus other unknown factors may be interfering with their normal compensation for high hemoglobin -oxygen affinity, and increased blood viscosity may have become a burden. Prudence dictates that before embarking on a regimen of chronic phlebotomy, one should be conservative and review the hematologic and physiologic findings at 6-month intervals during the first few years after diagnosis. In older patients, special attention should be directed to blood flow to the heart and central nervous system. Thrombosis has been reported in individuals with high-oxygen-affinity variants but a causal relationship has not been established. In an example of a patient with a high-oxygen-affinity hemoglobin with concurrent β-thalassemia, treatment with hydroxyurea reduced the PCV from 61% to 39%, increased HbF from 3.6% to 30%, and P50 from 6 to 10 mm Hg.20 This case should not be a recommendation for routine treatment of erythrocytosis due to high-oxygen-affinity hemoglobins with cytostatic agents.
Low-oxygen-Affinity Hemoglobins
Hemoglobin variants with reduced affinity for oxygen are in many respects the converse of high-oxygen-affinity variants. About half as many low-oxygen-affinity variants have been described compared to high-oxygen-affinity variants. They are expressed in the heterozygote, with homozygosity likely to be embryonic lethal. A major clinical feature is anemia that, at times, is accompanied by cyanosis.
Pathophysiology
Alterations of critical molecular regions involved directly in the R-T transition result in the stabilization of the deoxy T state or destabilization of the oxy R state. Low-oxygen-affinity hemoglobins deliver more O2 to the tissues per gram of hemoglobin, and this is reflected by an oxygen-hemoglobin dissociation curve shifted toward the right of normal and an increase in P50. When hemoglobin has a right-shifted or low-affinity curve, the difference between oxygen binding in the lungs at PO2 levels of 100 mm Hg and unloading in the tissues at 40 mm Hg can be twice as great as the differences in a hemoglobin with a normal oxygen equilibrium curve. Patients with a moderately right-shifted oxygen equilibrium curve (P50 between 35 and 55 mm Hg) may be mildly anemic, but some individuals with very right-shifted oxygen equilibrium curves (P50 ˜80) are not anemic. A right-shifted oxygen equilibrium curve leads to an increase in the synthesis of 2,3-DPG and a decrease in its destruction.
Diagnosis
The first report of a low-oxygen-affinity hemoglobin was Hb Kansas (HBB asn102thr), which presented with asymptomatic cyanosis without anemia or hemolysis.21 Detection of a lowoxygen-affinity hemoglobin is part of the differential diagnosis of patients with cyanosis (see later). Before undertaking extensive diagnostic procedures in cases of cyanosis that are not clearly the result of cardiovascular or pulmonary disease, fractionating hemoglobin by HPLC and measuring blood P50 is advisable. A search for low-affinity hemoglobins as an explanation for anemia without cyanosis is less compelling, but if other investigations prove fruitless, unexplained normocytic anemia without reticulocytosis might be evaluated by measuring P50.
A simple bedside test can distinguish cyanosis resulting from low-oxygen-affinity hemoglobins or cardiopulmonary cyanosis from that occurring with methemoglobinemia, M hemoglobins, or sulfhemoglobinemia. When blood from carriers of low-oxygen-affinity hemoglobins or patients with cardiopulmonary disease is exposed to ambient oxygen, it will turn from purple-greenish to bright red. In contrast, blood of patients with methemoglobinemia, sulfhemoglobinemia, or M hemoglobins will remain abnormally colored.
Clinically apparent cyanosis is observed only in carriers of low-oxygen-affinity variants with greatly right-shifted curves and where the variant comprises a substantial portion of the hemolysate. Cyanosis is present from birth in some low-oxygen-affinity hemoglobins due to α-globin chain mutants. In carriers of β-globin chain mutants, cyanosis may appear from the middle to the end of the first year of life as γ-globin gene expression and HbF synthesis wanes and is replaced by β-globin gene expression and HbA. Neonatal cyanosis has been associated with γ-globin variants that have low oxygen affinity.22 Globin gene sequencing is the sole means of definitive diagnosis.
Clinical Features
Three low-oxygen-affinity variants have been described at β102. Hb Kansas, the best-studied variant, has a whole-blood P50 of ˜70 mm Hg, decreased co-operativity, and a normal Bohr effect.21 The β102 asn residue is invariant among β-globin chains and participates in the only hydrogen bond between asn 102 and asp 94 across the α1β2 interface in oxyhemoglobin. This bond is broken when the molecule assumes the T state. The new thr residue is incapable of forming this bond, and low oxygen affinity results from destabilization of the R conformer. The changes induced by this substitution at the α1β2 interface allow Hb Kansas to dissociate into αβ dimers, the near opposite of the high-oxygen-affinity Hb Chesapeake.
Hb Beth Israel (HBB asn102ser) was found in a patient with cyanosis of the fingers, lips, and nail beds.23 The P50 was 88 mm Hg, and arterial blood was only 63% saturated despite a normal pO2. The hemolysate also had a low oxygen affinity and a normal Bohr effect. Erythrocyte 2,3-DPG was mildly elevated. The molecular mechanism of reduced oxygen affinity is the same as for Hb Kansas, although the defect may be more disruptive locally, because the serine side chain is shorter than that of threonine.
Hb Bologna (HBB lys61met) is informative because it was present as a compound heterozygote with β0-thalassemia and comprised 90% of the hemolysate.24 Adults were neither cyanotic nor anemic despite having a P50 of 37.6 mm Hg. During gestation, the high concentration of HbF makes it doubtful that this mutation would have an effect on fetal development.
Hb Bruxelles (HBB phe42del) is a deletion of the most conserved amino acid residue of hemoglobin.25, 26 Phenylalanine residues at β41 and β42 are conserved in all normal mammalian non-α-globin chains and are indispensable for the structural integrity and oxygen-binding functions of the molecule. From age 4 years, the index case of Hb Bruxelles had severe hemolytic anemia and cyanosis, requiring blood transfusion once. Later in life, her hemoglobin concentration stabilized at 10 g/dL. Reasons for this “switch” of phenotype are unknown. Other mutations of β41 and β42, which are predominately unstable hemoglobins, are discussed later.
Treatment
Treatment is not needed for these variants. The importance of early diagnosis is to avoid unnecessary work-up and to alleviate concern for the patient and family.
UNSTABLE HEMOGLOBINS
The unstable hemoglobins result from globin chain mutations that cause hemoglobin tetramer instability and intracellular precipitation of its globin subunits. These intraerythrocytic precipitates are detectable by supravital staining and appear as globular aggregates called Heinz bodies. These inclusions reduce the life of the erythrocyte by binding to the membrane, decreasing cell deformability, and increasing membrane permeability. The resultant hemolytic disorder is sometimes called congenital Heinz body hemolytic anemia. Heinz bodies and hemolysis also occur with certain hereditary erythrocyte enzyme deficiencies (see Chapter 28). More than 140 unstable variants of both the β– and α-globin chains with widely varying clinical severity have been reported, almost always as heterozygotes for the mutation, although some homozygous cases have been reported. The major clinical features are anemia, reticulocytosis, pigmenturia, and splenomegaly.
Pathophysiology
The pathophysiology of unstable hemoglobins relates to the specific mutations leading to altered heme-globin interaction, the process of Heinz body formation, and destruction of red cells containing denatured hemoglobin.
Mutations That Alter Heme-Globin Interaction
Mutations that change the primary structure (amino acid sequence) of globin, depending on the substitution and its location, can alter the secondary structure (α-helical), the tertiary structure (folding of the globin chain), or the quaternary structure (interactions within the hemoglobin tetramer). The mechanisms that can lead to this hemoglobin instability are listed in Table 35.2.
Heme-globin interactions are vital for oxygen delivery but also contribute to molecular stability and intracellular solubility. For example, introduction of a charged amino acid residue into the heme pocket, a site normally formed by residues with nonpolar side chains, results in hemoglobin instability (e.g., Hb Bristol). Mutations involving residues that interact directly with heme, such as those near the (F8) proximal histidine that reacts with heme-iron (e.g., Hb Köln), are associated with hemoglobin instability. Also, mutations associated with nontyrosine substitutions of the (E7) distal histidine (e.g., Hb Zürich) cause molecular instability. An interesting effect of this is that the ligand-binding properties of iron are changed, and Hb Zürich has a much higher affinity for CO.27, 28
Disruption of the secondary structure reduces subunit solubility and is often a result of the introduction of proline residue that cannot be accommodated into the α-helix except in its first two positions. α-Helices comprise ˜70% of a globin subunit and must be folded into a globin motif. Introduction of water into the molecule destroys its stability, and this can be caused by substitution of a charged residue, for example, alanine, for a nonpolar residue, such as proline (e.g., Hb Brockton).29
TABLE 35.2 SITES OF GLOBIN MUTATION ASSOCIATED WITH UNSTABLE HEMOGLOBINSa | |||||
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Loss of intersubunit contact hydrogen bonds or salt bridges in the α1β1 contact area will interfere with hemoglobin quaternary structure and also reduce stability. Dissociation of α1β1 contacts normally does not occur in red cells, whereas dissociation of α1β2 contact does take place. Dissociation of α1β1 dimers into monomers is normally minimal, as it generates methemoglobin and consequent instability. Dissociation of chains along the α1β1 contact generates α– and β-globin chains that uncoil, loosening their heme-globin interaction and favoring methemoglobin formation. Mutations affecting the α1β1 interface tend to be more unstable than those affecting the α1β2 contact. Examples of unstable hemoglobin mutations that are due to decreased α1β1 contact include Hb Philly,30 Hb Peterborough,31 and Hb Stanmore.32
α-Hemoglobin-stabilizing protein (AHSP) binds free α-globin chains, protecting them from precipitation (see Chapter 6). In vitro studies suggest that the impaired interaction of AHSP with α-globin variants when the mutation lies in the molecular sites where ASHP binds α-globin might affect the stability of the variant. In these studies, recombinant Hb Groen Hart (HBA pro119ser), Hb Diamant (HBA pro119leu), and α-globin termination mutants had impaired interactions with AHSP. These observations suggest an additional mechanism for unstable α-globin variants.33, 34
Heinz Body Formation
Heme loss is inhibited by maintaining heme iron in the reduced ferrous (Fe2+) state by the action of methemoglobin reductases and detoxification of oxygen radicals. Therefore, dimerization and the dispersion and precipitation of free heme is minimized. Hemoglobin dimers autoxidize and lose heme more readily than tetramers. Generation of methemoglobin increases the thermoinstability of hemoglobin, suggesting that the pathways and events accompanying the conversion of ferrous to ferric heme are important for hemoglobin stability.
Heinz bodies are the product of hemoglobin denaturation. First suggested to be heme-depleted globin chains, these inclusions were subsequently identified as hemichromes, derivatives of ferric hemoglobin that have the sixth coordination position occupied by a ligand provided by the globin. Hemichromes are generated when heme is dissociated from the heme pocket and rebinds elsewhere in the globin after the α– or the β-chains have denatured. Irreversible hemichromes are a stage in the formation of Heinz bodies (also see Chapter 6). Membranes prepared from the red cells of patients with Hb Köln (HBB val98met) who have had splenectomies contain aggregates composed of disulfide-linked spectrin, Band 3, globin, and high-molecular-weight complexes composed in part of denatured spectrin.
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